The innate immune system is designed to guard against potentially dangerous infections, but recent studies also show that our immune system can become activated by "self-antigens" which are released during severe acute illnesses, such as trauma, pancreatitis or burns. Under these conditions, tissue damage results in widespread activation of the immune system in the absence of any identifiable infection (i.e., sterile inflammation), which is associated with a dramatic worsening of the patient's condition. Dendritic cells (DCs) are among the immune cells that first respond to antigens released from damaged cells, and new data from our laboratory indicates that mitochondrial components of the cell most vigorously activate DCs. In particular, unmethylated (CpG) mitochondrial DNA when combined with its usual binding partner, mitochondrial transcription factor A (TFAM), is shown to potently activate DCs. We hypothesize that the mitochondrial antigens TFAM and CpG DNA play a major role in the induction of DC activation in the context of acute cell damage. To test this hypothesis three major research aims are proposed: 1) We will determine if RAGE and TLR-9/MyD88 pathways independently contribute to DC activation by TFAM and CpG DNA, 2) we will determine the role of PI-3K/Akt signaling in promoting DC Type 1 interferon responses to TFAM and CpG DNA, 3) we will determine if the DNA-binding Pro-Lys-Arg sequence of TFAM protein is responsible for its pro- inflammatory biological activity. For these studies we will use human DCs and DCs obtained from genetically altered mice lacking RAGE, TLR-9, and MyD88. We will also determine if TFAM and CpG DNA are responsible for the activation of the immune response in mice. These experiments will investigate an antibody directed against TFAM, which may serve as a mechanism to block the activation of the immune system in response to acute cell death, which would have important applications for the treatment of critically ill animals and humans. PUBLIC HEALTH RELEVANCE: This project will determine the mechanisms linking cell and tissue damage to activation of the immune system, which is a common and potentially life-threatening complication in acutely ill patients, such as those suffering from trauma, pancreatitis or burn injuries. Recent studies in our laboratory indicate that dendritic cells, which represent a first line of defense of our immune system, are strongly activated in response to components of mitochondria, which are released from damaged cells. This project will focus on the mechanisms by which a particular mitochondrial protein, mitochondrial transcription factor A (TFAM), engages the immune system to promote inflammation. This project has important implications for understanding how cell and tissue damage, such as commonly occurs in many acute illnesses, can induce activation of the immune system, which is potentially harmful to the host.